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This version published online on January 29, 2004
Molecular Endocrinology, doi:10.1210/me.2003-0391
Molecular Endocrinology Vol. 0, No. 2004 200303911-
doi:10.1210/me.2003-0391
Copyright © 2004 by the Endocrine Society.
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Submitted on October 9, 2003
Accepted on January 19, 2004

Insulin Regulates PKC{beta}II Alternative Splicing in Multiple Target Tissues: Development of a Hormonally Responsive Heterologous Minigene

Niketa A. Patel, Hercules S. Apostolatos, Konrad Mebert, Charles E. Chalfant, James E. Watson, T. S. Pillay, Janet Sparks, and Denise R. Cooper*

Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612; The Research Service, James A. Haley Veterans Hospital, Tampa, Florida 33612; Department of Biochemistry, Virginia Commonwealth University, and Hunter Holmes McGuire Veterans Medical Center, Richmond, VA 23298-0614; Institute of Cell Signaling, University of Nottingham Medical School, Queen's Medical Center, Nottingham NG7 2UH, UK; University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642.

* To whom correspondence should be addressed. E-mail: dcooper{at}hsc.usf.edu.

Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the PKC{beta} isozymes and preferentially switches the expression to PKC{beta}II isozyme shown to have a crucial role in glucose uptake, cellular proliferation and differentiation. We have developed an insulin responsive PKC{beta}II heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKC{beta}II exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKC{beta}II alternative splicing in five distinct cell types, i.e. L6 skeletal muscle, 3T3-L1 pre-adipocytes, HepG2 human hepatoma cells, A10 vascular smooth muscle cells and murine embryonic fibroblasts within 30 min of insulin stimulation. Sequential deletions of the flanking introns in the minigene demonstrated that insulin regulated elements within the 5' intron flanking the PKC{beta}II exon. Mutational studies indicated the SRp40 binding site promotes splice site selection. In these cases, splicing appears to be regulated by a PI-3 kinase signaling pathway since LY294002 and wortmannin, its specific inhibitors, blocked exon inclusion. Co-transfection with constitutively active Akt2 kinase mimicked insulin action. Signal-dependent regulation of splicing by insulin is unique from tissue-specific and developmentally regulated mechanisms previously reported, and serves as a prototype for studies of alternative splicing involving protein phoshorylation.
Key words: alternative splicing • hormonally-regulated • insulin • PKC{beta}




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